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Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins

Nature Cell Biology volume 13, pages 1344–1352 (2011)Cite this article

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Abstract

Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with numerous aggregation diseases. Several protein quality control mechanisms degrade non-native proteins by the ubiquitin–proteasome system. Here, we use quantitative mass spectrometry to demonstrate that heat-shock triggers a large increase in the level of ubiquitylation associated with misfolding of cytosolic proteins. We discover that the Hul5 HECT ubiquitin ligase participates in this heat-shock stress response. Hul5 is required to maintain cell fitness after heat-shock and to degrade short-lived misfolded proteins. In addition, localization of Hul5 in the cytoplasm is important for its quality control function. We identify potential Hul5 substrates in heat-shock and physiological conditions to reveal that Hul5 is required for ubiquitylation of low-solubility cytosolic proteins including the Pin3 prion-like protein. These findings indicate that Hul5 is involved in a cytosolic protein quality control pathway that targets misfolded proteins for degradation.

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Figure 1: Heat-shock stress induces protein misfolding and polyubiquitylation.
Figure 2: Heat-shock mainly affects cytosolic proteins.
Figure 3: HUL5 is required for the full ubiquitylation response and cell fitness after heat-shock.
Figure 4: Hul5 redistribution to the cytoplasm is important for its role in the heat-shock response.
Figure 5: HUL5 is essential for the ubiquitylation of proteins misfolded in the absence of SSA-chaperone activity and for the degradation of pulse-labelled misfolded polypeptides.
Figure 6: HUL5 is required for ubiquitylation of low-solubility cytosolic proteins.
Figure 7: Hul5 targets proteins that are specifically ubiquitylated in the low-solubility cellular fraction.

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Acknowledgements

The authors express gratitude to the following people for providing reagents: D. Finley (Harvard University, USA; HUL5 plasmids); J. Thorner (University of California, Berkeley, USA; anti-Pgk1 antibody); L. Howe (University of British Columbia, Canada; anti-H3 antibody); A. Chruscicki (University of British Columbia, Canada; Dynal-IgG); S. Jentsch (Max Planck Institute of Biochemistry, Germany; E2 double deletion strains); E. Craig (University of Wisconsin–Madison, USA; ssa1-45 strains); L. Conibear (University of British Columbia, Canada; TAP strains); R. Deshaies (California Institute of Technology, USA), C. Boone (University of Toronto, Canada) and M. Roberge (University of British Columbia, Canada) (deletion strains). We also thank P. Kaiser for comments on the manuscript, J. Gsponer for discussion, T.M. laboratory members for their encouragement and discussion and the following people for their support: L. Foster and N. Stoynov (mass spectrometry analysis); A. Chang (microscopy); P. Hieter and J. Stoepel (plate reader analysis); E. Jan (35S labelling and Odyssey). T.M. is supported by a grant from the Canada Institutes of Health Research (CIHR) and is a CIHR New Investigator. V.M. is supported by a grant from the CIHR.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Centre for High-Throughput Biology, University of British Columbia, 2125 East Mall, Vancouver, British Columbia V6T 1Z4, Canada

    Nancy N. Fang, Alex H. M. Ng & Thibault Mayor

  2. Wine Research Centre, University of British Columbia, 2205 East Mall, Vancouver, British Columbia V6T 1Z4, Canada

    Vivien Measday

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Contributions

N.N.F. and T.M. conceived the project and designed experiments; N.N.F. carried out experiments; A.H.M.N. designed and carried out the SSA1 analysis and developed reagents; V.M. designed and carried out part of the localization analysis; N.N.F., A.H.M.N., V.M. and T.M. analysed the data; N.N.F., V.M. and T.M. wrote the manuscript.

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Correspondence to Thibault Mayor.

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Fang, N., Ng, A., Measday, V. et al. Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins. Nat Cell Biol 13, 1344–1352 (2011). https://doi.org/10.1038/ncb2343

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